Markers of metabolic endotoxemia as related to metabolic syndrome in an elderly male population at high cardiovascular risk: a cross-sectional study

The prevalence of metabolic syndrome (MetS) is rapidly increasing in the western world. Its health consequences includes a twofold increased risk of developing cardiovascular disease (CVD), a sevenfold increased risk of developing diabetes mellitus type 2 (T2DM) and a 1.5-fold increased risk of all-cause mortality [1, 2].

There is no uniform definition of MetS, which essentially is a testimony of its complexity. MetS is a cluster of clinical and biochemical conditions which are often found to coexist, thus indicating a common pathophysiological background. The common denominators are central obesity, insulin resistance/glucose intolerance, dyslipidemia and hypertension [3], also known as Norman Kaplan’s “deadly quartet” [4].

The underlying pathophysiology remains unclear and is widely debated. There is, however, compelling evidence that MetS is associated with chronic low-grade inflammation, typically demonstrated by increased levels of C-reactive protein (CRP) and pro-inflammatory cytokines like interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFα) compared to subjects without MetS [5, 6]. Abdominal adipose tissue seems to be an important source of this inflammatory response, and the size and composition of this compartment have been shown to correlate well with the amount of circulating pro-inflammatory cytokines [7].

Translocation of parts of the gut microbiome, and in particular endotoxins or lipopolysaccharides (LPS) to the systemic circulation, has been proposed to be an early trigger of inflammation and subsequent cardiovascular risk [8]. An increase in plasma LPS can occur in healthy individuals after a high fat meal, partly due to co-transportation over the gut wall together with dietary fat by incorporation in triglyceride-rich chylomicrons [9, 10]. Leakage through dysfunctional tight-junctions have also been suggested [11]. Obese individuals tend to have higher levels of circulating LPS, both in fasting conditions and in the postprandial phase [10], and circulating levels of LPS have been reported to correlate with abdominal obesity and glycemic control [12]. In experimental human studies, chronic LPS exposure has been shown to promote systemic insulin resistance and adipose tissue related inflammation [13].

To communicate with the innate immune system, LPS binds to the LPS-binding protein (LBP), which is pivotal for the binding of CD14 and transfer to the Toll like receptor (TLR) 4 complex [14]. Further activation of NF-κB and interferon regulatory factors induces transcription of pro-inflammatory mediators. Blockage of LBP or CD14-binding seems to attenuate the inflammatory effect of LPS in animal studies [15, 16]. Circulating levels of LBP have also been reported to correlate with abdominal obesity and glycemic control [12, 17].

We hypothesize that microbial translocation may contribute to the inflammatory state associated with MetS. We therefore explored any association between circulating levels of LBP and soluble CD14 (sCD14) and the presence of MetS, its components and hyperglycemia. We also explored any association to systemic inflammation.

Baseline characteristics and laboratory data of the total study population are shown in Table 1. As displayed, 38% met the criteria of MetS. Within the criteria of MetS, 88% fulfilled the hypertension criterion, 29% the waist criterion, 40% the hypertriglyceridemia criterion, 10% the low HDL criterion and 56% the impaired fasting glucose criterion. Within the individuals who did not fulfill the definition of MetS (n = 300), 56% had two criteria fulfilled, corresponding to 35% of the total population. Only 6% of these, corresponding to 4% of the total population, had no criteria. Furthermore, there were 16% diabetics, 33% current smokers, 29% with previously diagnosed cardiovascular disease (CVD) and 31% were treated for hypertension (Table 1).

In the present study, concentrations of LBP and sCD14 did not differ significantly in the subjects with MetS vs no MetS although a numerically higher level of LBP was found in the MetS group. We observed, however, a trend towards increasing risk of MetS through quartiles of LBP, in line with previous reports that have shown significant associations between MetS and LBP [22, 23]. The prevalence of comorbidities and the frequent use of medications in both the MetS and the no MetS group, could explain the limited differences in our study, hence the no MetS group cannot be looked upon as a true control group as they may share some of the same metabolic disturbances as seen in MetS. This is further emphasized by the fact that only 6% of the individuals without MetS, did not fulfill any of the MetS criteria and more than 50% had two criteria fulfilled. There are also limited knowledge about how medications affect leakage of LPS from the gut and the expression of sCD14 and LBP. Previous studies have excluded patients on antidiabetics and statins, or use of such medications have not been properly reported [23, 24].

Looking at anthropometric measures in our study population, none of the markers correlated with BMI, however, a significant association was observed between LBP and waist circumference. Central obesity is thought to play a vital role and even accelerate the metabolic and hormonal disturbances observed in this syndrome. The amount of intraabdomial fat has been shown to correlate strongly with serum concentration of LPS as well as bacterial DNA found in intraabdominal fat samples [12]. Thus, although correlations were weak, our finding supports an association and potential link between central obesity and metabolic endotoxemia.

Dyslipidemia in MetS is typically characterized with low levels of HDL and high triglyceride levels. LPS has been shown to be inversely correlated with HDL, thus one could expect an equal relationship between LBP and HDL [25]. In our study, LBP was however, not significantly correlated with HDL or with any of the other lipoproteins in the population as a whole, also when excluding patients on statins, which is known to influence inflammation [26]. In the group with MetS alone, triglycerides were significantly correlated to sCD14.

Glucometabolic parameters are also central in MetS. Hyperglycemia has independently been associated with increased leakage of gut microbial content [27]. We did however, not find any significant differences in LBP nor sCD14 levels between the different ranges of glucose.

Of the glucometabolic parameters, only serum insulin showed a weak positive correlation with LBP in the whole population, while TG/HDL ratio correlated weakly, but significantly to sCD14 in the MetS group. We did not observe any correlations between our markers of gut leakage and HOMA, which is probably due to lack of correlation to fasting glucose. Experimental studies with chronic LPS exposure, typically show decreased insulin sensitivity [13, 28], and several studies have shown correlation between insulin resistance and LBP in humans [17, 29]. Antidiabetics such as sulfonylureas and metformin, whose central mechanisms of action are to stimulate pancreatic beta cells production of insulin and increase glucose sensitivity, respectively, could mask a potential association. However, in our study, the frequency of such drugs was low.

We could also show that the proposed markers of gut leakage, LBP and sCD14, correlate well with downstream mediators of systemic inflammation in the total cohort. For the group with MetS, the correlations were somewhat weaker, which may be due to the reduction in sample size. We have previously shown that IL-6, IL-18, CRP and TNFα, all were significantly higher in the group with MetS compared to those without [21].

LBP is essentially an acute phase protein secreted primarily by hepatocytes and the transcription of LBP is mainly stimulated by IL-1, IL-6 and LPS [30, 31]. Free or membrane-bound LPS is the main ligand of LBP, although it can also bind to other lipopeptides such as Lipoteichoic acid [32], thus not being LPS exclusive. sCD14 can also interact with other pathogen associated molecular patterns (PAMPs) and TLRs, thus not being specific for the LPS–LBP complex [33].

Our population is rather homogenous in terms of being only males and with a narrow age span between 65 and 75 years. Consequently, we were not able to examine differences between sexes and different age groups. Concentrations of LBP are known to increase with increasing age [23].

We have used LBP and sCD14 as surrogate markers of gut related endotoxemia. As stated, both LBP and sCD14 do not exclusively bind to LPS. Conversely, other PAMPs are able to activate the immune system independent of LBP and sCD14. As a result, we are only able to highlight parts of the gut-related activation of the innate immune system.

Furthermore, we did not register daily alcohol intake. Excessive alcohol intake is associated with increased levels of LPS and LBP in serum [34]. Endotoxemia has also been suggested as an important mechanism behind alcohol induced fatty liver. Thus, this may be an important confounder that we are not able to adjust for.

In our study, multiple analyses were conducted using two dependent variables, thereby adding to the risk of Type I errors. However, we decided not to perform Bonferroni correction, because we look upon this study as a hypothesize-generating study, and believe that a correction would be too strict in this case.

To conclude, our study cohort of elderly men cannot confirm that levels of LBP or sCD14 contribute significantly to the low grade inflammation in subjects with MetS. Nevertheless, there was a trend for increased prevalence of MetS with increasing quartiles of LBP which seems to be mainly driven by central obesity. Furthermore, we reaffirm that both LBP and sCD14 are associated with systemic inflammation, indicating a role of the innate immune system in MetS.